The present invention relates generally to devices and methods for the culturing of cells on electrode surfaces, and more specifically to culture dishes for the culturing of adherent normal cell monolayer cultures wherein they may be subjected to an electric field or discharge and to the use of transparent thin film electrodes conducive to cell adhesion for use in conjunction with such applications.
The term "petri dish" as used herein refers to that "shape/function", familiarly known to those skilled in the art of cell culture as a petri dish. The term "pepetri dish" is used herein to refer to a planar electrode petri-type dish according to one embodiment of the invention. The terms "fluid", "media" and "medium" as used herein refer to such materials as may be used for the culturing and or suspension of cell cultures. Similarly, the terms "electroporation fluid" or "electroporation medium" refer to those groups of materials and solutions that may be used in the process of electroporation.
The expressions "cell/s", "culture/s", and "cell culture/s" as used herein include those operations starting from the process of "plating", and up to and including the stage known as "confluency"; i.e. from a starting point of one cell to the point at which the culture surface is entirely covered by a monolayer of cells and substantially no further cell division occurs in normal cells. This range is meant to include known cell culturing techniques, such as synchronous culturing, and those that make up the wide range that would be used in micro-biology, neuro-biology, pharmacology and other related fields of endeavour.
The term "exogenous materials" as used herein refers to macromolecules such as DNA, RNA, proteins, plasmids, and other such materials that may be of interest for introduction into a living cell.
Adherent cells/cultures as used herein refers to those types of cells that are anchorage-dependent for growth.
Diverse biological responses to electric fields, both applied and endogenous, continue to motivate experimental searches for mechanisms of electromagnetic interactions with cells. Jaffe, L. F. (1979) Control of development by ionic currents. In Membrane Transduction Mechanisms. R. A. Cone and J. E. Downing editors. Raven, N.Y. 199-231, has shown that cell development is effected by an electric field, while Borgens, R. B., J. W. Vanable, Jnr., and L. F. Jaffe (1977) Bioelectricity and Regeneration. I. Initiation of frog limb, describe the effect of electric fields on cell regeneration. Many other basic cellular functions, including motility and receptor regulation are also modulated by applied external electric fields. In addition, cell membrane permeabilization and fusion have been effected by applied fields (see Zimmerman, U., and J. Vienken (1982) Electric field-induced cell-to-cell fusion. J. Membr. Biol. 67:165-182; Tessie, J., V. P. Knutson, T. Y. Tsong, and M. D. Lane (1982) Electric pulse-induced fusion of 3t3 cells in monolayer culture. Science (Wash. D.C.). 216:537-538; and Potter, H., L. Wier, and P. Leder (1984) Enhancer-dependent expression of human K immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation).
Local perturbation of plasma membrane potentials provides a hypothetical mechanism for the interaction of applied electric fields with cells.
Thus, Canadian Patent No. 1,208,146 (Wong) describes a method of transferring genes into cells which comprises subjecting a mixture of the genes and the cells suspended in a liquid medium, and subjected to an electric field and electric discharge. U.S. Pat. No. 4,663,292 (Wong) discloses a method of transferring genes into cells and fusing cells, which comprises subjecting suspensions of genes and cells and cells to an electric discharge.
U.S. Pat. No. 4,695,547 (Hilliard et al) relates to a multi-welled tray that contains a suspension of cells and the foreign molecule, and wherein a ring-shaped metallic electrode configuration that does not interfere with visual observation by inverted microscope during the procedure is received from above within the well. U.S. Pat. 4,764,473 (Matschke et al) discloses a device for the electroporation and electrofusion of cells using a double helical metallic electrode configuration and is for the application of electric fields to cells in suspension.
U.S. Pat. No. 4,561,961 (Hofmann, see FIG. 3,) discloses an electrofusion apparatus wherein a sandwiched chamber containing the metallic electrodes may be placed in the microscope, while German Offen. 3,321,239 (Zimmermann et al) describes an electrofusion cell of very simple structure.
A large percentage of interest in mammalian cell lines lies in the group known is adhesion-dependent types. Most primary fibroblasts proliferate when attached to glass or plastic, but do not grow while in suspension. Adhesion dependent cells do not adhere to metallic surfaces. Studies into "anchorage dependance", a term that describes the inability of normal cells to grow unless attached to a substratum, have shown that cells do not enter the S-phase (i.e. the reproduction cycle of cell growth when DNA is undergoing replication) unless attached to an appropriate substratum. While significant work has been done towards understanding the interaction between cells and applied electric fields, this has been virtually restricted to single cells in suspension, and is therefore of very limited application in the study of the far more complex interplay between applied electric fields and cells in monolayer culture tissue, and virtually no work has been able to be done on cells while in the S-phase of growth.
Certain procedures such as electroporation and electrofusion require subjecting cell samples to electric fields. Treating the cells entails trypsinizing the cells, rinsing, suspending them in an appropriate fluid carrying the foreign molecule, in most cases chilling the resulting suspension, subjecting the cells to the electric field, rinsing, and then re-plating them.
Current cell electroporation equipment requires the significant expenditure of relatively high voltage and current, to the point that cooling is often a concern. This is due to the relative inefficiencies in mutually dependent system parameters, such as the number of cells per unit volume of electrolytic media; the conductivity and composition of the electrolytic media; the voltage being applied; the time period of pulse, the location of the cell relative to the electrode; and the area of membrane surface being presented in relation to the electrodes (spherical) are all factors which require attention when using suspensions and does not account for any change incurred in the cell or to the membrane as a result of being put into suspension. The exogenous material (DNA/RNA code, etc.) that it would be of interest to have expressed as a measurable cell function upon successful integration is open to cyto-enzymatic attack until integrated or metabolized. Normal cell function is recovered after when the electrical inducing poration is no longer present, but progression of normal cell functions such as replication can not occur until sufficient adhesion is regained to induce a growth signal. A significant period of time may elapse between the poration process and potential integration and has an affect on efficiency. Depending upon the cell condition after transmembrane inductance of exogenous material and the type of material introduced, transient, stable or no expression may be expected. Due to the adverse conditions imposed in the process to date, a large percentage of cells die, some give a transient expression and fewer still exhibit a permanent stable expression.
Because of this large number of cell deaths, a shift in the base population occurs since a first level of selection (survivors vs non-survivors) has been made which further complicates matters.
It is the purpose of this invention to overcome many of the above stated drawbacks as currently known in the art and to significantly advance the state of the art.